Wild Animals in Captivity: An Analysis of Parasite Biodiversity and Transmission among Animals at Two Zoological Institutions with Different Typologies.

We have conducted a 10-year-long coprological study of the animals housed in two zoological institutions (ZooAquarium and Faunia, Madrid, Spain) to assess the parasite biodiversity, prevalence, and their relation with host class, diet, and enclosure type (soil type and level of isolation from wild fauna). A total of 4476 faecal samples from 132 mammal species and 951 samples from 86 avian species were examined. The results indicated that only 12.8% of avian species had parasites at least once during the study period, whereas 62.1% of mammal species tested positive. Predominantly, protists (Entamoeba, flagellates, and ciliates) and nematodes (mainly Trichuris) were identified in the findings. Carnivorous species were primarily infected by nematodes, while herbivorous and omnivorous species were mainly infected by protists. The number of infected herbivorous and omnivorous species was significantly greater than carnivorous species. Differences were observed based on soil type (artificial, natural, mixed) and isolation level (isolated/accessible), but these differences were not statistically significant. Several parasites (Entamoeba spp., Giardia spp., Balantidoides coli, Trichuris spp.) could potentially be transmitted between humans and some mammals and birds. Regular animal analyses and a personnel health program in the institutions would minimise transmission risks between zoo animals, wildlife, and humans.

Prevalence of Toxoplasma gondii in Endangered Wild Felines (Felis silvestris and Lynx pardinus) in Spain.

The wildcat (Felis silvestris) and the Iberian lynx (Lynx pardinus) are important species in Spain, considered as near-threatened and endangered, respectively. Both can be infected by Toxoplasma gondii, a parasite that can cause morbidity and mortality in transplacentally-infected or immunocompromised mammals. The data on the prevalence of this parasite in wild populations of these species in Spain are outdated. The objective of this study was to update information and evaluate the role of these felines in parasite epidemiology and the potential impact of the parasite on their conservation. Blood and fecal samples were collected from captured animals, as well as the tongue, diaphragm, and spleen, from animals killed in road accidents in central Spain. An indirect fluorescent antibody test (IFAT) was used to detect parasite antibodies in serum, microscopy and molecular analysis were used to detect oocysts in feces, and molecular analysis was used to determine the existence of tissue cysts. Seroprevalence was 85% in wildcats and 45% in lynx, and parasite DNA was detected in the feces of one wildcat and in tissue samples from 10 wildcats and 11 Iberian lynxes. These results highlight the epidemiological importance and high risk of T. gondii infection in animals and humans in the studied areas. Considering feline susceptibility to infection, monitoring programs are needed to assess the health status of wild felines.

Human-Borne Pathogens: Are They Threatening Wild Great Ape Populations?

Climate change and anthropic activities are the two main factors explaining wild great ape habitat reduction and population decline. The extent to which human-borne infectious diseases are contributing to this trend is still poorly understood. This is due to insufficient or fragmented knowledge on the abundance and distribution of current wild great ape populations, the difficulty obtaining optimal biological samples for diagnostic testing, and the scarcity of pathogen typing data of sufficient quality. This review summarises current information on the most clinically relevant pathogens of viral, bacterial, parasitic, and fungal nature for which transmission from humans to wild great apes is suspected. After appraising the robustness of available epidemiological and/or molecular typing evidence, we attempt to categorise each pathogen according to its likelihood of truly being of human origin. We further discuss those agents for which anthroponotic transmission is more likely. These include two viral (Human Metapneumovirus and Respiratory Syncytial Virus), one bacterial (diarrhoeagenic Escherichia coli), and two parasitic (Cryptosporidium spp. and Giardia duodenalis) pathogens. Finally, we identify the main drawbacks impairing research on anthroponotic pathogen transmission in wild great apes and propose research lines that may contribute to bridging current knowledge gaps.

Chromosome-scale Echinococcus granulosus (genotype G1) genome reveals the Eg95 gene family and conservation of the EG95-vaccine molecule.

Cystic echinococcosis is a socioeconomically important parasitic disease caused by the larval stage of the canid tapeworm Echinococcus granulosus, afflicting millions of humans and animals worldwide. The development of a vaccine (called EG95) has been the most notable translational advance in the fight against this disease in animals. However, almost nothing is known about the genomic organisation/location of the family of genes encoding EG95 and related molecules, the extent of their conservation or their functions. The lack of a complete reference genome for E. granulosus genotype G1 has been a major obstacle to addressing these areas. Here, we assembled a chromosomal-scale genome for this genotype by scaffolding to a high quality genome for the congener E. multilocularis, localised Eg95 gene family members in this genome, and evaluated the conservation of the EG95 vaccine molecule. These results have marked implications for future explorations of aspects such as developmentally-regulated gene transcription/expression (using replicate samples) for all E. granulosus stages; structural and functional roles of non-coding genome regions; molecular 'cross-talk' between oncosphere and the immune system; and defining the precise function(s) of EG95. Applied aspects should include developing improved tools for the diagnosis and chemotherapy of cystic echinococcosis of humans.

Cyst detection and viability assessment of Balantioides coli in environmental samples: Current status and future needs.

The ciliate Balantioides coli is a human enteric parasite that can cause life-threatening infections. It is a food- and waterborne parasite, with cysts being the infective stage. Despite its importance as a potential pathogen, few reports have investigated its presence in environmental samples, and some issues need attention including i) The accuracy of B. coli identification. In most cases, the protozoa is identified only by its morphological traits, which can be identical to those from other parasitic ciliates of animals. Genetic analysis of cysts recovered from environmental samples is necessary for species confirmation. In addition, genetic methods used with faecal samples need to be adequately validated with environmental matrices. ii) The methodology for searching this parasite in environmental samples. The protocols include an initial phase to isolate the cysts from the matrix followed by a second phase in which concentration procedures are usually applied. The methods may be valid but are not standardised and differences between studies could affect the results obtained. iii) The areas that needs further research. The development of genetic identification methods and standardised analytical protocols in environmental samples are required, as well as the assessment of viability and infectivity of B. coli cysts. The development of axenic culture systems will boost research on this parasite.

Presence and genetic diversity of enteric protists in captive and semi-captive non-human primates in côte d'Ivoire, Sierra Leone, and Peru.

Little information is currently available on the occurrence and genetic diversity of pathogenic and commensal protist species in captive and semi-captive non-human primates (NHP) resident in zoological gardens or sanctuaries in low- and medium-income countries. In this molecular-based study, we prospectively collected individual faecal samples from apparently healthy NHP at the Abidjan Zoological Garden (AZG) in Côte d'Ivoire, the Tacugama Sanctuary (TS) in Sierra Leone, and the Quistococha Zoological Garden (QZG) in Peru between November 2018 and February 2020. We evaluated for the presence of pathogenic (Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi, Balantioides coli) and commensal (Entamoeba dispar, Troglodytella abrassarti) protist species using PCR methods and Sanger sequencing. Giardia duodenalis was the most prevalent species found (25.9%, 30/116), followed by Blastocystis sp. (22.4%, 26/116), and E. dispar (18.1%, 21/116). We detected E. bieneusi (4.2%, 1/24) and T. abrassarti (12.5%, 3/24) only on NHP from AZG. Cryptosporidium spp., E. histolytica, and B. coli were undetected at the three sampling sites investigated here. Sequence analyses revealed the presence of zoonotic sub-assemblages BIII (n = 1) in AZG and BIV (n = 1) in TS within G. duodenalis. We identified Blastocystis subtype ST3 (100%, 6/6) in AZG, ST1 (80.0%, 12/15), ST2 (6.7%, 1/15), and ST3 (13.3%, 2/15) in TS, and ST2 (80.0%, 4/5) and ST3 (20.0%, 1/5) in QZG. The only E. bieneusi isolate detected here was identified as zoonotic genotype CAF4. Our PCR-based data indicate that potentially pathogenic protist species including G. duodenalis, Blastocystis sp., E. bieneusi, and B. coli are present at variable rates in the three NHP populations investigated here. The identification of zoonotic genotypes within these species indicates that human-NHP transmission is possible, although the extent and directionality of these events need to be elucidated in future molecular surveys.

Enteric protists in wild western chimpanzees (Pan troglodytes verus) and humans in Comoé National Park, Côte d'Ivoire.

The western chimpanzee (Pan troglodytes verus), a subspecies of the common chimpanzee, is currently listed as Critically Endangered. Human-driven habitat loss and infectious diseases are causing dramatic chimpanzee population declines and range contractions that are bringing these primates to the brink of extinction. Little information is currently available on the occurrence of diarrhoea-causing enteric protist species in chimpanzees in general, and in western chimpanzees in particular, or on the role of humans as a potential source of these infections. In this prospective molecular epidemiological study, we investigated the presence, genetic variability, and zoonotic potential of enteric protists in faecal samples from western chimpanzees (n = 124) and humans (n = 9) in Comoé National Park, Côte d'Ivoire. Parasite detection and genotyping were conducted by using polymerase chain reaction (PCR) and Sanger sequencing. The protist species found in the chimpanzee samples were Entamoeba dispar (14.5%), Blastocystis sp. (11.3%), Giardia duodenalis (5.8%), Troglodytella abrassarti (2.5%) and Cryptosporidium hominis (0.8%). The protist species found in the human samples were G. duodenalis (22.2%) and Blastocystis sp. (11.1%). Entamoeba histolytica, Enterocytozoon bieneusi, and Balantioides coli were undetected in both chimpanzee and human samples. Sequence analyses revealed the presence of Blastocystis subtype (ST) 1 (alleles 4 and 8) and ST3 (allele 24) in chimpanzees, and ST3 (allele 52) in humans. ST1 allele 8 represents a chimpanzee-adapted Blastocystis genetic variant. Cross-species transmission of pathogenic enteric protists between chimpanzees and humans might be possible in Comoé National Park, although the frequency and extent of zoonotic events remain to be fully elucidated.

Molecular Detection and Characterization of Intestinal and Blood Parasites in Wild Chimpanzees (Pan troglodytes verus) in Senegal.

Wild chimpanzee populations in West Africa (Pan troglodytes verus) have dramatically decreased as a direct consequence of anthropogenic activities and infectious diseases. Little information is currently available on the epidemiology, pathogenic significance, and zoonotic potential of protist species in wild chimpanzees. This study investigates the occurrence and genetic diversity of intestinal and blood protists as well as filariae in faecal samples (n = 234) from wild chimpanzees in the Dindefelo Community Nature Reserve, Senegal. PCR-based results revealed the presence of intestinal potential pathogens (Sarcocystis spp.: 11.5%; Giardia duodenalis: 2.1%; Cryptosporidium hominis: 0.9%), protist of uncertain pathogenicity (Blastocystis sp.: 5.6%), and commensal species (Entamoeba dispar: 18.4%; Troglodytella abrassarti: 5.6%). Entamoeba histolytica, Enterocytozoon bieneusi, and Balantioides coli were undetected. Blood protists including Plasmodium malariae (0.4%), Trypanosoma brucei (1.3%), and Mansonella perstans (9.8%) were also identified. Sanger sequencing analyses revealed host-adapted genetic variants within Blastocystis, but other parasitic pathogens (C. hominis, P. malariae, T. brucei, M. perstans) have zoonotic potential, suggesting that cross-species transmission between wild chimpanzees and humans is possible in areas where both species overlap. Additionally, we explored potential interactions between intestinal/blood protist species and seasonality and climate variables. Chimpanzees seem to play a more complex role on the epidemiology of pathogenic and commensal protist and nematode species than initially anticipated.

Long-Term Preservation and Storage of Faecal Samples in Whatman® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis.

Preservation and conservation of biological specimens, including faecal samples, is a challenge in remote areas or poor-resource settings where the cold chain cannot be maintained. This study aims at evaluating the suitability of filter cards for long-term storage of faecal samples of animal and human origin positive to the diarrhoea-causing protozoan parasites, Giardia duodenalis and Cryptosporidium hominis. Three commercially available Whatman® Filter Cards were comparatively assessed: the FTA® Classic Card, the FTA® Elute Micro Card, and the 903 Protein Saver Card. Human faecal samples positive to G. duodenalis (n = 5) and C. hominis (n = 5) were used to impregnate the selected cards at given storage (1 month, 3 months, and 6 months) periods and temperature (-20 °C, 4 °C, and room temperature) conditions. Parasite DNA was detected by PCR-based methods. Sensitivity assays and quality control procedures to assess suitability for genotyping purposes were conducted. Overall, all three Whatman® cards were proven useful for the detection and molecular characterisation of G. duodenalis and C. hominis under the evaluated conditions. Whatman® cards represent a simple, safe, and cost-effective option for the transportation, preservation, and storage of faecal samples without the need of the cold chain.

Occurrence and Genetic Diversity of Protist Parasites in Captive Non-Human Primates, Zookeepers, and Free-Living Sympatric Rats in the Córdoba Zoo Conservation Centre, Southern Spain.

Little information is currently available on the epidemiology of parasitic and commensal protist species in captive non-human primates (NHP) and their zoonotic potential. This study investigates the occurrence, molecular diversity, and potential transmission dynamics of parasitic and commensal protist species in a zoological garden in southern Spain. The prevalence and genotypes of the main enteric protist species were investigated in faecal samples from NHP (n = 51), zookeepers (n = 19) and free-living rats (n = 64) by molecular (PCR and sequencing) methods between 2018 and 2019. The presence of Leishmania spp. was also investigated in tissues from sympatric rats using PCR. Blastocystis sp. (45.1%), Entamoeba dispar (27.5%), Giardia duodenalis (21.6%), Balantioides coli (3.9%), and Enterocytozoon bieneusi (2.0%) (but not Troglodytella spp.) were detected in NHP. Giardia duodenalis (10.5%) and Blastocystis sp. (10.5%) were identified in zookeepers, while Cryptosporidium spp. (45.3%), G. duodenalis (14.1%), and Blastocystis sp. (6.25%) (but not Leishmania spp.) were detected in rats. Blastocystis ST1, ST3, and ST8 and G. duodenalis sub-assemblage AII were identified in NHP, and Blastocystis ST1 in zookeepers. Giardia duodenalis isolates failed to be genotyped in human samples. In rats, four Cryptosporidium (C. muris, C. ratti, and rat genotypes IV and V), one G. duodenalis (assemblage G), and three Blastocystis (ST4) genetic variants were detected. Our results indicate high exposure of NHP to zoonotic protist species. Zoonotic transmission of Blastocysts ST1 was highly suspected between captive NHP and zookeepers.

Intestinal Protists in Captive Non-human Primates and Their Handlers in Six European Zoological Gardens. Molecular Evidence of Zoonotic Transmission.

We assessed the occurrence, genetic diversity, and zoonotic potential of four protozoan (Cryptosporidium spp., Entamoeba histolytica, Entamoeba dispar, Giardia duodenalis), one stramenopile (Blastocystis sp.), one microsporidia (Enterocytozoon bieneusi), and two ciliate (Balantioides coli, Troglodytella abrassarti) intestinal parasite or commensal protist species in captive non-human primates (NHP) and their zookeepers from six European zoological gardens in France (n = 1), Germany (n = 1), and Spain (n = 4). Faecal samples from NHP (n = 454) belonging to 63 species within 35 genera and humans (n = 70) were collected at two sampling periods in each participating institution between October 2018-August 2021. Detection and species identification was accomplished by PCR and Sanger sequencing of the ssu rRNA and/or ITS genes. Sub-genotyping analyses using specific markers were conducted on isolates positive for G. duodenalis (gdh, bg, tpi) and Cryptosporidium spp. (gp60). Overall, 41.0% (186/454) and 30.0% (21/70) of the faecal samples of NHP and human origin tested positive for at least one intestinal protist species, respectively. In NHP, Blastocystis sp. was the most prevalent protist species found (20.3%), followed by G. duodenalis (18.1%), E. dispar (7.9%), B. coli and T. abrassarti (1.5% each), and Cryptosporidium spp. and E. bieneusi (0.9% each). Occurrence rates varied largely among NHP host species, sampling periods, and zoological institutions. The predominant protist species found in humans was Blastocystis sp. (25.7%), followed by Cryptosporidium spp. (2.9%), E. dispar (1.4%), and G. duodenalis (1.4%). Sequencing of PCR-positive amplicons in human and/or NHP confirmed the presence of Cryptosporidium in six isolates (C. hominis: 66.7%, C. parvum: 33.3%), G. duodenalis in 18 isolates (assemblage A: 16.7%, assemblage B: 83.3%), Blastocystis in 110 isolates (ST1:38.2%, ST2:11.8%, ST3: 18.2%, ST4: 9.1%, ST5: 17.3%, ST8: 2.7%, ST13: 0.9%), and E. bieneusi in four isolates (CM18: 75.0%, Type IV: 25.0%). Zoonotic transmission events involving Blastocystis ST1-ST4 were identified in four zoological institutions. Zoonotic transmission of C. hominis was highly suspected, but not fully demonstrated, in one of them. Monitoring of intestinal protist species might be useful for assessing health status of captive NHP and their zookeepers, and to identify transmission pathways of faecal-orally transmitted pathogens.

Balantioides coli.

Balantioides coli (=Balantidium coli) is the only ciliate that parasitizes humans. Pigs are the main reservoir. Other species, as camels, cattle, donkey, sheep and goat have been also proposed as reservoirs for human infections. The parasite has a direct life cycle, being transmitted by the faecal-oral route. This type of cycle and the large number of host species imply an important potential for zoonotic transmission of the parasite. Infections are most commonly found in tropical and temperate regions, with prevalence up to 100% in pigs; high prevalence values have been also recorded in some non-human primates and camels. In humans, prevalence is usually under 10% in the population at risk. The main epidemiological factors involved in the transmission of this parasite include close contact with pigs, lack of basic sanitation infrastructures (water supply, wastewater disposal) and hygiene. Individual health status, intestinal microbiota and diet are also important for the onset of the infection. Outbreaks caused by this parasite are rare; those reported to date were related to poor hygienic conditions or to catastrophic natural disasters. Balantioides coli infections can be asymptomatic and symptomatic, which can be chronic (with intermittent diarrhoea), or acute (a dysenteric form which can be life-threatening). Efective treatments include tetracycline, iodoquinol and 5-nitroimidazole compounds (metronidazole, secnidazole). The main effective individual preventive measure is the use of disinfected water for drinking and other uses. Adequate water supply infrastructures, proper disposal of wastewater and animal faeces, and regular monitoring programs on farms will help limit transmission.

Morphological and molecular characterization of a new ciliate Nyctotheroides grimi n. sp. (Armophorea, Clevelandellida) from Chinese frogs.

A new species of clevelandellid ciliate, Nyctotheroides grimi n. sp., is described from the frog Fejervarya limnocharis. Light and scanning electron microscopy were used for the morphological studies, and the DNA encoding the SSU rRNA gene (SSU rDNA) and the ITS1-5.8S subunit rRNA-ITS2 region (ITS) were sequenced for genetic comparisons and phylogenetic analysis. The main distinctive morphological feature is a knob-like projection in the left-posterior end; other differential characters are the cell size, the length of the oral groove and the shape of the infundibulum. Nyctotheroides grimi possess an apical suture line in the left and right side of the anterior end and in the left side of the caudal end. In the phylogenetic analyses, the new species engroups with other Nyctotheroides species forming a monophyletic group. The high similarity in the SSU rDNA and ITS sequences between Nyctotheroides species suggests a relative recent divergence. The genetic data and the different host range support the separation of Nyctotheroides and Nyctotherus; however the morphological criterion based on the presence (in Nyctotheroides)/absence (in Nyctothterus) of an apical kinetal suture line should be modified to consider the presence of kinetal suture lines in the apical and/or the caudal left side in Nyctotheroides.

Morphological description of Opalina obtrigonoidea Metcalf, 1923 (Heterokonta, Opalinea) from Duttaphrynus melanostictus and evaluation of the ITS region as a suitable genetic marker for inter-species identification in Opalina.

The redescription of Opalina obtrigonoidea Metcalf, 1923, collected from the rectum of the toads Duttaphrynus melanostictus, is presented in this paper based on detailed morphological information and molecular data. Our results revealed that O. obtrigonoidea varies greatly in body dimensions. Its morphological characteristics allow its differentiation from Opalina undulata. Surprisingly, we sequenced its SSU rDNA-ITS1-5.8S rDNA-ITS2-LSU rDNA (5' end) and found the SSU rDNA of O. obtrigonoidea is nearly identical to that of O. undulata. However, there are differences in both the ITS1 and ITS2 regions that allow their distinction and confirm the morphological differences. Our results indicate that O. obtrigonoidea and O. undulata are closely related species in which morphological and genetic markers have evolved at different speeds. Due to this, the SSU rDNA gene may not be a valid marker for inter-species identification in Opalina, but the ITS is a valid marker for differentiating species in this genus.

Genetic identification of the ciliates from greater rheas (Rhea americana) and lesser rheas (Rhea pennata) as Balantioides coli.

The ciliate species Balantioides coli can be cross-transmitted between humans and several animal species. Usually harmless, sometimes it can be pathogenic and cause the death of the host. In birds, B. coli has been confirmed in ostriches by genetic analysis, but the identification from South American greater rheas (Rhea americana) and lesser rheas (Rhea pennata pennata) is tentative. Since these species are reared for commercial purposes and for reintroduction into the wild, it is necessary to elucidate whether the ciliate from rheas is B. coli to minimize health risks for humans and for other domestic and wild animals. Individual parasite cells are collected from Argentinean isolates of reared greater rheas and of wild and reared lesser rheas, and their ITS region was PCR amplified; the cloning products were sequenced and compared with sequences available in public databases. The results have shown that several sequence types are expressed at the same time in the parasite cells, and all correspond to B. coli, confirming the possibility of cross-transmission of the parasite between wild and reared South American rheas and several mammal species and humans.

Endoparasitism of Greek ostriches: First report of Entamoeba struthionis and Balantioides coli.

Ostrich farming is a worldwide practice and an internationally developing industry. Among challenges in livestock production are the parasitic infections. The present study aimed to the investigation of the gastrointestinal parasites biota and prevalence in ostriches raised in different areas of Greece. A total of 141 clinically healthy ostriches originating from four different localities of Greece were coprologically examined for parasites of the gastrointestinal tract. Coprological examination revealed a considerably high rate of infection (65.9%) with protozoa; however, no helminths (trematodes, cestodes and nematodes) were detected. In detail, cysts of Entamoeba struthionis have been found in 57.4% of the examined birds. Moreover, 39.0% of sampled ostriches harboured cysts of Balantioides coli (syn. Balantidium coli), while oocysts of Cryptosporidium sp. were detected at a low percentage (2.1%). Partial sequences of the small subunit rRNA (16S rRNA) gene and the ITS region were amplified from pooled Entamoeba and Balantioides positive samples, respectively, confirming for the first time the presence of Entamoeba struthionis and Balantioides coli in ostriches in Greece. Some of these parasitoses require attention as they may affect productivity performance of the animals in commercial ostrich farming and possibly pose disease risk for livestock and humans.

Redescription of Opalina triangulata (Heterokonta, Opalinea) from Fejervarya limnocharis based on morphological and molecular data.

Opalinids are a large group of anaerobic protists, mainly inhabiting the cloacae of amphibians (frogs and toads). The classification of this group has not been fully resolved, because of a lack of molecular information. Here, we give a redescription of Opalina triangulata Metcalf, 1923, collected from the rectum of the frog Fejervarya limnocharis, based on detailed morphological and molecular data. Our phylogenetic analyses confirmed the monophyly of Opalinata. Within it, Opalinea were monophyletic with O. triangulata and O. undulata as well as two Protoopalina species grouping together. Karotomorpha and Proteromonas did not group together confirming the paraphyly of Proteromonadea. Meanwhile, the ITS2 secondary structural similarities as well as G-C content revealed greater similitudes between Opalina species and P. lacertae than with Blastocystis hominis, which is in accordance with their position as sister clades in the SSU rDNA-based phylogenies.

Are molecular tools clarifying or confusing our understanding of the public health threat from zoonotic enteric protozoa in wildlife?

Emerging infectious diseases are frequently zoonotic, often originating in wildlife, but enteric protozoa are considered relatively minor contributors. Opinions regarding whether pathogenic enteric protozoa may be transmitted between wildlife and humans have been shaped by our investigation tools, and have led to oscillations regarding whether particular species are zoonotic or have host-adapted life cycles. When the only approach for identifying enteric protozoa was morphology, it was assumed that many enteric protozoa colonized multiple hosts and were probably zoonotic. When molecular tools revealed genetic differences in morphologically identical species colonizing humans and other animals, host specificity seemed more likely. Parasites from animals found to be genetically identical - at the few genes investigated - to morphologically indistinguishable parasites from human hosts, were described as having zoonotic potential. More discriminatory molecular tools have now sub-divided some protozoa again. Meanwhile, some infection events indicate that, circumstances permitting, some "host-specific" protozoa, can actually infect various hosts. These repeated changes in our understanding are linked intrinsically to the investigative tools available. Here we review how molecular tools have assisted, or sometimes confused, our understanding of the public health threat from nine enteric protozoa and example wildlife hosts (Balantoides coli - wild boar; Blastocystis sp. - wild rodents; Cryptosporidium spp. - wild fish; Encephalitozoon spp. - wild birds; Entamoeba spp. - non-human primates; Enterocytozoon bieneusi - wild cervids; Giardia duodenalis - red foxes; Sarcocystis nesbitti - snakes; Toxoplasma gondii - bobcats). Molecular tools have provided evidence that some enteric protozoa in wildlife may infect humans, but due to limited discriminatory power, often only the zoonotic potential of the parasite is indicated. Molecular analyses, which should be as discriminatory as possible, are one, but not the only, component of the toolbox for investigating potential public health impacts from pathogenic enteric protozoa in wildlife.

Analysis of nad2 and nad5 enables reliable identification of genotypes G6 and G7 within the species complex Echinococcus granulosus sensu lato.

The larval stages of tapeworms in the species complex Echinococcus granulosus sensu lato cause a zoonotic disease known as cystic echinococcosis (CE). Within this species complex, genotypes G6 and G7 are among the most common genotypes associated with human CE cases worldwide. However, our understanding of ecology, biology and epidemiology of G6 and G7 is still limited. An essential first step towards this goal is correct genotype identification, but distinguishing genotypes G6 and G7 has been challenging. A recent analysis based on complete mitogenome data revealed that the conventional sequencing of the cox1 (366 bp) gene fragment mistakenly classified a subset of G7 samples as G6. On the other hand, sequencing complete mitogenomes is not practical if only genotype or haplogroup identification is needed. Therefore, a simpler and less costly method is required to distinguish genotypes G6 and G7. We compared 93 complete mitogenomes of G6 and G7 from a wide geographical range and demonstrate that a combination of nad2 (714 bp) and nad5 (680 bp) gene fragments would be the best option to distinguish G6 and G7. Moreover, this method allows assignment of G7 samples into haplogroups G7a and G7b. However, due to very high genetic variability of G6 and G7, we suggest to construct a phylogenetic network based on the nad2 and nad5 sequences in order to be absolutely sure in genotype assignment. For this we provide a reference dataset of 93 concatenated nad2 and nad5 sequences (1394 bp in total) containing representatives of G6 and G7 (and haplogroups G7a and G7b), which can be used for the reconstruction of phylogenetic networks.

Oropharyngeal Trichomonosis Due to Trichomonas gypaetinii in a Cinereous Vulture ( Aegypius monachus) Fledgling in Spain.

A juvenile Cinereous Vulture ( Aegypius monachus) fledgling was found disorientated on the roof of a building in Madrid City, Spain, in October 2016. A veterinary examination revealed multiple plaques distributed throughout the oropharyngeal cavity. Lesions were located under the tongue and at the choanal slit, hard palate, and esophagus opening and ranged from 2 to 7 mm, coalescing in areas up to 2 cm, with a yellowish color of the surface. Motile trichomonad trophozoites were detected in fresh wet mount smears from the lesions. Sequence analysis of the internal transcribed spacer (ITS)1/5.8S/ITS2 and small subunit ribosomal RNA confirmed that Trichomonas gypaetinii was the etiologic agent. Microbiologic cultures did not reveal any pathogenic bacteria or fungi. The animal recovered successfully after treatment with metronidazole and trimethoprim-sulfamethoxazole and was later released in a suitable habitat. Avian trichomonosis lesions caused by T. gypaetinii have not been reported.